Growth factors are polypeptides which stimulate a wide variety of biological responses (e.g. DNA synthesis, cell division, expression of specific genes, etc.) in a defined population of target cells. A variety of growth factors have been identified, including the transforming growth factor beta family (TGF-βs), epidermal growth factor and transforming growth factor alpha (the TGF-αs), the platelet-derived growth factors (PDGFs), the fibroblast growth factor family (FGFs) and the insulin-like growth factor family (IGFs), which includes IGF-I and IGF-II. Many growth factors have been implicated in the pathogenesis of cancer.
IGF-I and IGF-II (the “IGFs”) are related in amino acid sequence and structure, with each polypeptide having a molecular weight of approximately 7.5 kilodaltons (kDa). IGF-I mediates the major effects of growth hormone, and is thus the primary mediator of growth after birth. IGF-I has also been implicated in the actions of various other growth factors, since the treatment of cells with such growth factors leads to increased production of IGF-I. In contrast, IGF-II is believed to have a major role in fetal growth. Both IGF-I and IGF-II have insulin-like activities (hence their names), and are mitogenic (stimulate cell division).
IGF-I has been found to stimulate the growth of cells from a number of different types of cancer (Butler et al., 1998 Cancer Res. 58(14):3021-3027; Favoni R E, et al., 1998, Br. J. Cancer 77(12): 2138-2147). IGF-I has additionally been found to exert anti-apoptotic effects on a number of different cell types, including tumor cells (Giuliano M, et al., 1998 Invest Ophthalmol. Vis. Sci. 39(8): 1300-1311; Zawada W M, et al., 1998, Brain Res. 786(1-2): 96-103; Kelley K W, et al., 1998, Ann. N.Y. Acad. Sci. 840: 518-524; Toms S A, et al., 1998, J. Neurosurg. 88(5): 884-889; Xu F, et al., 1997, Br. J. Haematol. 97(2): 429-440). Prospective studies have implicated IGF-I as a risk factor for cancers of the prostate, breast, and colon, while IGFBP-3, the major circulatory binding protein for IGFs, appears to have a protective effect (10-12, 28, 29). A variety of other observations further support the idea that the relative balance of IGFBP-3 to other IGF-binding proteins (notably IGFBP-2) is somehow instrumental in the control of tumor cell growth, both in vitro and in vivo (7-9). Recent evidence also suggests that IGFBP-3 may play a central role in the growth (13-17) and apoptosis (14) of tumor cells in an IGF-independent manner.
Approximately half of the 1.3 million patients diagnosed with cancer each year in the U.S. have (or will be at risk for) systemic disease. Chemotherapy is the most common therapeutic approach for these patients (34). Most chemotherapeutic agents are effective primarily against dividing cells, and myelosuppression is often the dose-limiting toxicity. Chemical agents fall into several categories and have different mechanisms of action but, at effective doses, most have side-effects which seriously impact the patient's quality of life. doxorubicin (ADRIAMYCIN®), irinotecan (CPT-11), paclitaxel (TAXOL®), cisplatin, tamoxifen, methotrexate and 5-fluorouracil are popular agents used to treat a variety of cancers, sometimes in combination. In addition to myelosuppression, gastrointestinal effects, mucositis, alopecia, and (in the case of doxorubicin) cardiac toxicities are also observed with these agents (34). Clearly, it would be of interest to find ways to make tumor cells selectively sensitive to these chemical agents.
Almost all IGF circulates in a non-covalently associated complex of IGF-I, insulin-like growth factor binding protein 3 (IGFBP-3) and a larger protein subunit termed the acid labile subunit (ALS), such that very little free IGF-I is detectable. The ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF-binding activity and appears to bind only to the IGF/IGFBP-3 complex (Baxter et al., J. Biol. Chem. 264(20):11843-11848, 1989), although some reports suggest that IGFBP-3 can bind to rat ALS in the absence of IGF (Lee et al., Endocrinology 136:4982-4989, 1995). The ternary complex of IGF/IGFBP-3/ALS has a molecular weight of approximately 150 kDa and has a substantially increased half-life in circulation when compared to binary IGF/IGFBP-3 complex or IGF alone (Adams et al., Prog. Growth Factor Res. 6(2-4):347-356; presented October 1995, published 1996). This ternary complex is thought to act “as a reservoir and a buffer for IGF-I and IGF-II preventing rapid changes in the concentration of free IGF” (Blum et al. (1991), “Plasma IGFBP-3 Levels as Clinical Indicators” in MODERN CONCEPTS OF INSULIN-LIKE GROWTHFACTORS, pp. 381-393, E. M. Spencer, ed., Elsevier, N.Y.). While there is essentially no excess (unbound) IGFBP-3 in circulation, a substantial excess of free ALS does exist (Baxter, J. Clin. Endocrinol. Metab. 67:265-272, 1988).
How IGFBP-3 mediates its cellular effects is not well understood, although there is indirect evidence to suggest that it mediates some of the effects of p53, a well-characterized tumor suppressor (Ferry et al., (1999) Horm Metab Res 31(2-3):192-202). IGFBP-3 is mobilized to the nucleus of rapidly growing cells (Schedlich, et al., (1998) J. Biol. Chem. 273(29):18347-52; Jaques, et al., (1997) Endocrinology 138(4):1767-70). A useful step toward defining the functional interactions of IGFBP-3 would be to identify protein domains involved in the ability of IGFBP-3 to specifically bind a surprisingly large array of intracellular and extracellular targets. Known targets include: IGF-I, IGF-II, insulin (under some conditions), acid-labile subunit (ALS), plasminogen, fibrinogen, transferrin, lactoferrin, collagen Type Ia, prekallikrein, RXR-alpha, viral oncoproteins, heparin, specific proteases, cellular receptors, a number of intracellular targets identified in two-hybrid screens, and components of the nuclear localization transport machinery (Mohseni-Zadeh and Binoux (1997) Endocrinology 138(12):5645-8; Collett-Solberg, et al. (1998) J. Clin. Endocrinol Metab. 83(8):2843-8; Rajah, et al. (1995) Prog. Growth Factor Res. 6(2-4):273-84; Fowlkes and Serra (1996) J. Biol. Chem. 271:14676-14679; Campbell, et al. (1999) J. Biol Chem. 274(42):30215-21; Durham, et al. (1999) Horm Metab Res 31(2-3):216-25; Campbell, et al. (1998) Am J Physiol. 275(2Pt 1):E321-31).
IGFBP-3 has three major domains, roughly corresponding to exons 1, 2 and 3+4 of the IGFBP-3 gene, respectively. The C-terminal domain of IGFBP-3 (Domain 3), which contains sequences homologous to a motif found in CD74 (invariant chain) and a number of other proteins, appears to be involved in IGFBP-3's ability to interact with serum, extracellular matrix, and cell surface components. Peptides made to sequences in this region have previously been shown to interfere with the binding of IGFBP-3 to a number of its known ligands, including RXR-alpha, transferrin, ALS, plasminogen, fibrinogen and pre-kallikrein (Liu, et al, J. Biol. Chem. 275: 33607-13, 2000; Weinzimer, et al, J. Clin. Endocrinol. Metab. 86: 1806-13, 2001; Campbell, et al, Am. J. Physiol. 275: E321-31, 1998; Campbell, et al, J. Biol. Chem. 274: 30215-21, 1999; Firth, et al, J. Biol. Chem. 273: 2631-8, 1998). However, to date, IGFBP-3-derived peptides have not been shown to be sufficient for selective, high-affinity binding to any of these ligands.
This region of the molecule has also been implicated in nuclear translocation, but the mechanism by which IGFBP-3 is internalized into target cells is not well understood (Schledlich, et al, J. Biol. Chem. 273: 18347-52, 1998; Jaques, et al, Endocrinology 138: 1767-70, 1997). A recently described mutant in which Domain 3 residues 228-232 of IGFBP-3 have been substituted with the corresponding residues from IGFBP-1 (a closely related protein) shows impaired binding to ALS, RXR-alpha, and plasminogen (Campbell, et al. (1998) Am. J. Physiol. 275(2 Pt 1):E321-31; Firth, et al. (1998) J. Biol. Chem. 273:2631-2638). Specific proteolysis of IGFBP-3 under certain physiological conditions such as pregnancy and critical illness can lead to altered binding and release of its IGF ligand. The binary complex of IGFBP-3 with IGF-I or IGF-II (both growth factors bind IGFBP-3, with similar affinities) can extravasate across endothelial junctions to the intercellular milieu where IGFBP-3 can interact specifically with glycosaminoglycans, specific proteases, and cell-surface proteins. Research reports have referred to the presence of a C-terminal domain in IGFBP-3 that can inhibit IGFBP-4 proteolysis (Fowlkes, et al, J. Biol. Chem. 270: 27481-8, 1995; Fowlkes, et al, Endocrinology 138: 2280-5, 1997). However, the exact location of this putative protease inhibitor domain has not yet been described. IGFBP-4 proteolysis is a key event in a number of biological processes, including pregnancy, post-angioplasty smooth muscle cell growth, bone formation, and ovarian follicular dominance (Byun, et al, J. Clin. Endocrinol. Metab. 86: 847-54, 2001; Bayes_Genis, et al, Arterioscler. Thromb. Vasc. Biol. 21: 335-41, 2001; Miyakoshi et al, Endocrinol. 142: 2641-8, 2001; Conover, et al, Endocrinol. 142: 2155, 2001; Rivera, et al, Biol. Reprod. 65: 102-11, 2001).
It should be noted that, while IGFBP-3 is the most abundant of the IGF binding proteins (“IGFBPs”), at least five other distinct IGFBPs have been identified in various tissues and body fluids. Although these proteins bind IGFs, they originate from separate genes and have distinct amino acid sequences. Unlike IGFBP-3, other circulating IGFBPs are not saturated with IGFs. IGFBP-3 and IGFBP-5 are the only known IGFBPs which can form the 150 kDa ternary complex with IGF and ALS. The IGF-binding domain of IGFBP-3 is thought to be in the N-terminal portion of the protein, as N-terminal fragments of the protein isolated from serum retain IGF binding activity. However, some of the other IGFBPs have also been suggested for use in combination with IGF-I as therapeutics.
In addition to its role as the major carrier protein for IGF in serum, IGFBP-3 has been recently shown to have a number of different activities. IGFBP-3 can bind to an as-yet unidentified molecule on the cell surface, where it can inhibit the activity of exogenously-added IGF-I (Karas et al., 1997, J. Biol. Chem. 272(26):16514-16520). Although the binding of IGFBP-3 to cell surfaces can be inhibited by heparin, the unidentified cell surface binding molecule is unlikely to be a heparin-like cell surface glycosaminoglycan, because enzymatic removal of heparin glycosaminoglycans has no effect on IGFBP-3 cell surface binding (Yang et al., 1996, Endocrinology 137(10):4363-4371). It is not clear if the cell surface binding molecule is the same or different than the IGFBP-3 receptor that was identified by Leal et al. (1997, J Biol. Chem. 272(33):20572-20576), which is identical to the type V transforming growth factor-beta (TGF-β) receptor.
IGFBP-3, when used alone in in vitro assays, has also been reported to promote apoptosis. Interestingly, IGFBP-3 has been shown to promote apoptosis in cells with and without functional type 1 IGF receptors (Nickerson et al., 1997, Biochem. Biophys. Res. Comm. 237(3):690-693; Rajah et al., 1997, J. Biol. Chem. 272(18):12181-12188). However, there are conflicting reports as to whether apoptosis is induced by full length IGFBP-3 or a proteolytic fragment of IGFBP-3 (Rajah et al., ibid; Zadeh et al., 1997, Endocrinology 138(7):3069-3072). More recently, a wealth of unpublished data gathered in a number of laboratories fails to support some of the claims made in the above publications. In in vivo models tested to date, infused IGFBP-3 protein alone has showed mixed results in limiting tumor growth.
U.S. Pat. No. 5,681,818 claims the administration of IGFBP-3 for controlling the growth of somatomedin dependent tumors in the treatment of cancer. U.S. Pat. No. 5,840,673 also describes the indirect intracellular modulation of IGFBP-3 levels as a method for controlling tumor growth. U.S. Pat. No. 6,015,786 discloses the use of IGFBP-3 complexed with mutant IGF for the treatment of IGF-dependent tumors. However, each of these patents discloses a direct in vivo effect of administered IGFBP-3 protein on tumor growth. All of these patents envisages the use of intact IGFBP-3, including its IGF-binding domain. Numerous publications (Williams, et al., Cancer Res 60(1):22-7, 2000; Perks, et al., J Cell Biochem 75(4):652-64, 1999; Maile et al., Endocrinology 140(9):4040-5, 1999; Gill, et al., J Biol Chem 272(41):25602-7, 1997) further demonstrate combined effects of IGF binding proteins, radiation and ceramide on cultured cells. In one report (Portera et al, Growth Hormone & IGF Research 2000, Supplement A, S49-S50, 2000) IGFBP-3 combined with CPT-11 showed additive effects in a colon cancer model both in vivo and in vitro. All of the above studies were conducted using intact IGFBP-3, a multifunctional molecule capable of carrying IGFs (which are anti-apoptotic) to cells, while also capable of exerting IGF-independent pro-apoptotic effects of its own. Clearly it would be of interest to separate these two activities at the molecular level, but molecules exhibiting a desirable subset of the activities of intact IGFBP-3 have not been described.
IGF-I and IGFBP-3 may be purified from natural sources or produced by recombinant means. For instance, purification of IGF-I from human serum is well known in the art (Rinderknecht et al. (1976) Proc. Natl. Acad. Sci. USA 73:2365-2369). Production of IGF-I by recombinant processes is shown in EP 0 128 733, published in December of 1984. IGFBP-3 may be purified from natural sources using a process such as that shown in Baxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256-1261). Alternatively, IGFBP-3 may be synthesized by recombinantly as discussed in Sommer et al., pp. 715-728, MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS (E. M. Spencer, ed., Elsevier, N.Y., 1991). Recombinant IGFBP-3 binds IGF-I in a 1:1 molar ratio.
Topical administration of IGF-I/IGFBP-3 complex to rat and pig wounds is significantly more effective than administration of IGF-I alone (Id.). Subcutaneous administration of IGF-I/IGFBP-3 complex to hypophysectomized, ovariectomized, and normal rats, as well as intravenous administration to cynomolgus monkeys, “substantially prevents the hypoglycemic effects” of IGF-I administered alone (Id.).
The use of IGF/IGFBP-3 complex has been suggested for the treatment of a wide variety of disorders (see, for example, U.S. Pat. Nos. 5,187,151, 5,527,776, 5,407,913, 5,643,867, 5,681,818 and 5,723,441, as well as International Patent Applications Nos. WO 95/03817, WO 95/13823, and WO 96/02565. IGF-I/IGFBP-3 complex is also under development by Insmed Pharmaceuticals, Inc., as a treatment for several indications, including diabetes and recovery from hip fracture surgery.
For practitioners skilled in the art, the complex of IGF-I and IGFBP-3 is generally considered to be a different compound, and to have different biological effects, than IGFBP-3 alone.
While there are a large number of cytotoxic drugs available for the treatment of cancer, these drugs are generally associated with a variety of serious side effects, including alopecia, leukopenia, mucositis. Accordingly, there is a need in the art for cancer therapies that do not induce the serious side effects associated with conventional cytotoxic chemotherapy. One method for achieving this goal is to make target cells (such as tumor cells) selectively sensitive to cytotoxic drugs, thereby permitting the effective use of such drugs at lower doses not associated with serious side effects. A pro-apoptotic peptide derived from IGF-binding protein may be capable of hastening the apoptotic response of tumor cells to chemotherapeutic and other agents (see copending U.S. application titled “Method for Use of IGF-Binding Protein for Selective Sensitization of Target Cells In Vivo” by D. Mascarenhas, filed Sep. 18, 2001).
Other applications for IGFBP-3, IGFBP-derived peptides and related molecules of the invention may be envisaged including modulators or diagnostic reporters of inflammatory and invasive processes in cancer metastasis, tumor stromal activation, autoimmune diseases such as systemic lupus erythrematosis (SLE), multiple sclerosis, diabetes, ankylosing spondulitis, ulcerative colitis, Crohn's and other inflammatory bowel disease, arthritis, asthma and allergy, bone resorptive disease, proliferative disease, wound healing, ophthalmological diseases including retinopathies, fibrotic diseases, reproductive biology, atherosclerosis and other cardiovascular indications; research tools useful in genomics- and proteomics-related applications including high-throughput screening tools in drug discovery and other research programs, reagents and vectors capable of enhancing existing technologies for rapid expression and screening of new genetic sequences, gene therapy, diagnostics and nanotechnology applications; and in stem cell-related applications.
Numerous natural and pathological processes involve an “inflammatory-invasive” or “inflammatory-migratory” condition. Examples include invasive tumors, blastocyst/cytotrophoblast implantation, atherosclerotic plaque build-up, bone turnover, joint swelling in arthritic conditions, relapsing-remitting autoimmune conditions such as multiple sclerosis, SLE and others, proliferative retinal diseases and activation of airway epithelium in asthmatics. A common feature of these biological processes is the activated state of cell types participating in local cross-talk relevant to the disease condition. For example, invasive epithelial tumors generally include (in addition to the tumor cells themselves), activated stromal cells, microvascular epithelial cells and inflammatory immune cells. Interventions targeting any of these cell types might be expected to influence overall disease patterns dramatically. The inventor has unexpectedly found that IGFBP-3 and IGFBP-derived peptides preferentially trigger cell death/apoptosis in such activated cells, compared to the same cell types without activation. A corroborating observation is the dependence of the co-apoptotic effects on alpha-5 and beta-1 integrins, which are known to be preferentially displayed by activated and migrating cells (Boles, et al, 2000, Am. J. Physiol. Lung Cell Mol. Physiol. 278: L703-L712; Laukaitis, et al, 2001,. J. Cell Biol. 153: 1427-1440) and in bone marrow micrometastases from epithelial tumors (Putz, et al, 1999, Cancer Res. 59: 241-248).
It is important to distinguish these effects from those relating to abrogation of IGF-I-dependent proliferative effects. The literature is replete with mention of IGF-I dependent inflammatory processes such as psoriasis. For example, U.S. Pat. No. 5,929,040 teaches the use of inhibitors targeting the IGF-I receptor, thereby reducing skin inflammation. IGFBPs can reduce signaling through this receptor by binding and thereby sequestering IGF-I. However, the IGFBP-derived peptides of this invention do not bind IGF-I and are not believed to exert their effects via the IGF-I receptor.
A distinction should also be made between the present invention and U.S. Pat. No. 5,527,776 which reveals the use of intact IGFBP-3/IGF-I complex to treat subjects with immune deficiencies and anemias. The present invention uses non-IGF-I-binding fragments derived from IGFBP-3 alone, to treat conditions characterized by immune stimulation rather than deficiency.
Consequently, IGFBP-3, IGFBP-derived peptides and related molecules of the invention may be envisaged as modulators or diagnostic reporters of angiogenic, osteoclastogenic, atherogenic, invasive, metastatic, reproductive, arthritic, asthmatic, fibrotic, retinopathic, infective, inflammatory, neurodegenerative, stress-related, cell remodeling- or immortalization-related biological processes.
In particular, IGFBP-3-derived peptides or smaller derivative molecules as disclosed herein may be used as protease inhibitors, metal chelators, anti-proliferative, anti-metastatic or anti-angiogenic molecules. They may also be useful as plasma carrier agents, facilitators of binding to extracellular matrix components, targeting agents, transporters of large or small compounds into cells (cell internalization agents), affinity purification tags, screening tags, transcriptional or DNA-binding agents, cell-labeling agents, regulatory modulators, or as agents exhibiting any combination of the above properties. In particular, such derivative molecules may be derived from the CD74-homology domain sequence at the carboxy-terminus of IGFBP-3, and many of these activities have never been localized to this region of the IGFBP-3 molecule before. Peptides made to sequences in this region have previously been shown to interfere with the binding of IGFBP-3 to a number of its known ligands, including RXR-alpha, transferrin, ALS, plasminogen, fibrinogen and pre-kallikrein (Liu, et al, J. Biol. Chem. 275: 33607-13, 2000; Weinzimer, et al, J. Clin. Endocrinol. Metab. 86: 1806-13, 2001; Campbell, et al, Am. J. Physiol. 275: E321-31, 1998; Campbell, et al, J. Biol. Chem. 274: 30215-21, 1999; Firth, et al, J. Biol. Chem. 273: 2631-8, 1998). However, to date, IGFBP-3-derived peptides have not been shown to be sufficient for selective, high-affinity binding to any of these ligands.
The IGFBP-3-derived metal-binding domain peptides disclosed herein differ from previously disclosed IGFBP-3-derived molecules in a number of important ways, including their inability to bind IGF-I, their unique antigenicity, and the absence of the IGFBP-3 putative death receptor (P4.33) interaction domain of IGFBP-3 (so-called “mid-region”; amino acids 88-148). The P4.33 putative death receptor is described in International Patent Application No. WO 01/87238 (Genbank Accession Number BC031217; gi:21411477). For example, International Patent Application No. WO 02/34916 teaches the use of point mutants of IGFBP-3 in which the binding to IGF-I is impaired. However, the described molecules contain the mid-region of IGFBP-3 and would be expected to exert biological effects by interacting with the P4.33 putative receptor. International Patent Application No. WO 01/87238 teaches the use of P4.33 modulators for treating disease. The metal-binding peptides of the present invention do not include the P4.33 putative interaction domain (mid-region of IGFBP-3). U.S. Pat. No. 6,417,330 teaches the use of IGFBP-3 variants which are modified to be resistant to hydrolysis. Also disclosed are variant IGFBP-3s where the nuclear localization signal (NLS) in native IGFBP-3 is altered. Additionally, amino-terminally extended IGFBP-3s are disclosed which include a variety of N-terminal extensions. All of these molecules differ from the metal-binding domain peptides of the present invention in two important ways: They bind IGF-I and they contain the mid-region of IGFBP-3, believed to interact with the P4.33 putative death receptor. Some recent publications have described the use of IGFBP-3 peptides to treat cells in culture. The only peptides found to be active on breast cancer cells are derived from the mid-region of IGFBP-3 (McCaig, et al, 2002, Br. J. Cancer 86: 1963-1969; Perks, et al, Bioch. Biophys. Res. Comm. 294: 988-994, 2002). This region is not present in the sequence of the metal-binding domain peptides of this invention.
It should be noted that any reference to any patent, patent application, or publication in this Background section is not an admission that such patent, patent application, or publication constitutes prior art to the instant invention.